Posttranslational modifications (PTMs) play vital roles in expanding protein functional diversity and critically affect numerous biological processes (C T Walsh et al., Angew. Chem. Int. Ed. Engl. Vol. 44 pp. 7342-72, 2005). The availability of proteins with specific modifications at selected residues is essential for experimental strategies to investigate fundamental biological mechanisms. Methods to generate diverse native protein covalent modifications currently do not exist.
Genetic code expansion approaches are useful in producing recombinant proteins with specific modifications (K. Lang et al., Chem. Rev. Vol. 114, pp. 4764-4806, 2014; C C Liu et al., Annu. Rev. Biochem. Vol. 79, pp. 413-44, 2010), but rely on the availability of an orthogonal tRNA/tRNA synthetase pair for acylation of a specific non-canonical amino acid. Despite much technical progress, the creation of many important protein modifications (e.g., trimethyl lysine) is not yet feasible.
Cys-based strategies have been widely applied to generate protein conjugates or mimics of PTMs (E. V. Vinogradova et al., Nature. Vol. 526, pp. 687-691, 2015; M. D. Simon et al., Cell. Vol. 128. pp. 1003-1012, 2007; S, I. van Kasteren et al., Nature. Vol. 446, pp. 1105-09, 2007). However, the final products produced by such methods are PTM analogs whose value for searching out unidentified properties of the natural system may still be questionable (D. P. Nguyen et al, Chem. Biol. Vol. 17, pp. 1072-76, 2010).
Thus, despite such extensive efforts, synthetic approaches for many authentic PTMs are not available, as no C—C bond forming reactions have been successfully applied to protein modifications despite the prevalence of such reactions in organic chemistry.
The present inventors have developed a method of producing a site-specifically phosphorylated protein by use of a SepRS variant and an EF-Tu variant (U.S. Pat. No. 9,322,044, Korean Patent Application No. 10-2016-0053885). Still, this method has a limitation in that it enables only selective phosphorylation among many PTMs present in intracellular proteins.
Meanwhile, phosphoamino acids are known to be labile under alkaline conditions (Y. Oda et al., Nat. Bioechnol. Vol. 19, pp. 379-82, 2001).
Also, reactions that form carbon-carbon bonds using transition metals as catalysts for generation of diverse organic compounds have been reported in recent years (A. Postigo et al., Chem. Rev. Vol. 255. pp. 2991-3030, 2001; B. H. Lipshutz et al., J. Am. Chem. Soc. Vol. 134. pp. 19985-88, 2012).
Under this background, the present inventors have made extensive efforts to produce a site-specifically modified protein via new carbon-carbon bond formation for the first time. As a result, the inventors have found that, when a specific site in a target protein is marked with a phosphorylated amino acid, is activated under alkaline conditions, and then is conjugated with an organic halogen compound containing a desired modification moiety or chemical group in the presence of a transition metal under optimized reaction condition, a site-specifically modified protein having the same chemical and functional properties as that of authentic PTMs present in cells can be produced, thereby completing the present invention.
The information disclosed in the Background Art section is only for the enhancement of understanding of the background of the present invention, and therefore may not contain information that forms a prior art that would already be known to a person of ordinary skill in the art.